Culture method and identification method of rat alveolar cavity stem cells

Abstract

The invention provides a method for acquiring, culturing and identifying rat alveolar cavity stem cells, which is characterized by comprising the following steps of: selecting an SD adult rat to establish a rat alveolar cavity model, performing histological staining and immunohistochemical analysis, judging and acquiring an optimal time node of DSCs according to the expression condition of p75NTR, and identifying the rat alveolar cavity stem cells according to the optimal time node. Early healing tissues are dug in the third day when the rat tooth extraction nest model is established so as to obtain the DSCs. The rat alveolar cavity stem cells obtained by the method have better growth activity and proliferation potential, have no obvious difference in stem cell characteristics, cell phenotype stable expression and cell proliferation efficiency in an in-vitro continuous passage process, and have multidirectional differentiation potential of mesenchymal stem cells. And the proliferation activity, the clone formation ability and the osteogenic differentiation ability of the obtained and cultured rat alveolar cavity stem cells are superior to those of rat periodontal ligament stem cells.

Description

Technical Field

[0001] The invention belongs to the field of stem cell biotechnology and relates to the acquisition, cultivation and identification of rat alveolar stem cells. Background Art

[0002] Tissue engineering and regenerative medicine with mesenchymal stem cells as the core are developing rapidly. It is expected that the formation of cementum, periodontal ligament and alveolar bone complex with complex anatomical structure can be achieved by transplanting stem cells. Therefore, researchers at home and abroad have been committed to discovering stem cells that are more conducive to periodontal tissue regeneration. Patent Publication No. CN116531408A uses umbilical cord blood hematopoietic stem cells and osteogenic biomaterials to combine into tooth osteogenic or gingival regeneration materials and proves that umbilical cord blood stem cells can promote the growth of osteoblasts or promote bone production, but the osteogenic differentiation ability of umbilical cord blood hematopoietic stem cells is not shown; Patent Publication No. CN103305465A uses sox-2 gene to sort cranial neural crest-derived stem cells from the jaw process tissue of 11-12 day mouse embryos and identifies the stem cell phenotype and osteogenic differentiation ability. Because it comes from specific tissues in the early embryo, the material is limited; Patent Publication No. CN115920050A improves the level of autophagy in the inflammatory environment by regulating the expression of TMEM110 protein, and promotes the osteogenic regeneration ability of periodontal ligament stem cells. Although periodontal ligament stem cells are considered to be the ideal seed cells for periodontal regeneration in tissue engineering, they have limitations such as limited survival during long-term culture, low proliferation efficiency, difficulty in obtaining, limited quantity, and the need to consider individual differences in practical applications.

[0003] Patients who need to have their teeth extracted due to advanced periodontitis or orthodontic treatment will have their alveolar sockets regenerated after tooth extraction, and the sockets will gradually heal. Studies have shown that the healing alveolar socket contains many early healing tissues, which are composed of undifferentiated mesenchymal stem cells, blood vessels, connective tissue matrix and immature mineralized bone. These undifferentiated mesenchymal stem cells have strong biological activity and osteogenic regeneration ability, which increases the bone healing efficiency of the sockets and are called alveolar socket stem cells. In 2014, Nakajima et al. reported that they scraped healing tissue from beagle extraction sockets for stem cell culture and called the obtained stem cells DSCs. They also showed that such stem cells have multidirectional differentiation ability in vitro and can regenerate periodontal complex structure after combining with biological scaffold materials; and their P75NTR positive expression rate is as high as 63.0%, and their proliferation and migration abilities are higher than those of bone marrow mesenchymal stem cells.

[0004] DSCs have good biological properties and have high proliferation, self-renewal and multidirectional differentiation abilities when cultured in vitro. Compared with other types of stem cells, DSCs can be obtained from extracted wisdom teeth or other alveolar bones, which is easier to obtain and the quantity is considerable. Moreover, DSCs can be derived from the patient himself, so there is no problem of immune rejection. At the same time, because they come from the sockets of wisdom teeth that have fallen off naturally or need to be extracted, they are highly acceptable in terms of ethics. In summary, as a new source of dental stem cells, DSCs have heterogeneous composition, complex cell populations, and rich sources. They have their own unique advantages in tissue engineering and regeneration. They are a potential source of stem cells and have important application prospects for clinical treatment and tissue repair. Summary of the invention

[0005] The purpose of the present invention is to provide a method for obtaining and culturing rat alveolar stem cells.

[0006] The purpose of the present invention is achieved by the following measures:

[0007] A method for obtaining and culturing rat alveolar stem cells, characterized in that: SD adult rats are selected to establish a rat alveolar socket model, and early healing tissue is excavated on the third day after the rat extraction socket model is established to obtain DSCs. On the third day after the rat extraction socket model is established, the wound heals well and is filled with healing tissue with a soft texture and a certain shape, and it is easy to obtain sufficient early healing tissue in the alveolar socket at this time. HE staining and immunohistochemical staining were performed on the rat alveolar socket model on days 0, 1, 3, and 7 after establishment. The results showed that the alveolar socket was filled with fibrous connective tissue on the third day, and the DAB staining positive expression area was mainly scattered around the fibrous connective tissue in the center of the alveolar socket.

[0008] p75 neurotrophic factor receptor (p75NTR) is a low-affinity neurotrophic factor neurotrophic receptor that plays an important regulatory role in biological processes such as mesenchymal stem cell proliferation and differentiation. It can be used as a surface marker to select dental mesenchymal stem cells with stronger odontogenic potential. P75NTR may assist tissue reconstruction in the early stage of alveolar socket healing as a special medium and participate in biological outcomes such as DSCs proliferation and differentiation. The expression of p75NTR can be used to determine the optimal time point for obtaining DSCs.

[0009] The determination of the time point for obtaining the above-mentioned rat DSCs includes the following steps:

[0010] On the 0th, 1st, 3rd and 7th days after the establishment of the rat alveolar socket model, one rat was killed by anesthesia, and the left mandible of the modeled rat was dissected and made into paraffin sections for HE staining and immunohistochemical staining to observe the formation of alveolar socket healing tissue and the expression of P75NTR. The results of HE staining showed that immediately after tooth extraction and on the first day after tooth extraction, a large number of red blood cells were accumulated in the alveolar socket, and a small number of osteoclasts were seen at the bone edge; on the third day after tooth extraction, a small number of red blood cells were accumulated in the alveolar socket, and the alveolar socket was replaced by fibrous connective tissue; on the 7th day after tooth extraction, new bone cells were formed in the alveolar socket, the number increased and the arrangement was disordered, forming a large area of ​​bone callus, and fibrous connective tissue hyperplasia and a large number of osteoclasts were seen around it. Immunohistochemistry results showed that P75NTR expression was observed on the inner wall of the alveolar socket and the edge of the bone marrow foramen immediately after tooth extraction and on the first day after tooth extraction, but no staining was observed in the center of the alveolar socket; on the third day after tooth extraction, the DAB staining-positive expression areas were mainly scattered around the fibrous connective tissue in the center of the alveolar socket, and a small amount of staining was also observed on the inner wall of the alveolar socket; on the seventh day after tooth extraction, P75NTR was expressed in the fibrous connective tissue at the edge of the new trabeculae.

[0011] A method for obtaining and culturing rat alveolar stem cells comprises the following steps: obtaining an early alveolar healing tissue block, centrifuging and digesting the tissue to disperse the tissue into a cell suspension, wherein the method is characterized in that enzymatic digestion of the tissue block for 50 minutes can successfully digest the tissue fragments and the cell clumps to obtain a suspension.

[0012] The above-mentioned method for obtaining and culturing rat alveolar stem cells also includes the following steps: discarding the supernatant after centrifuging the above-mentioned suspension, adding complete culture medium, and resuspending the cells, characterized in that: the centrifugal speed is 800rpm, and the centrifugal time is 5 minutes, which can effectively remove cell precipitation, remove digestive enzymes and ensure cell activity.

[0013] The method for obtaining and culturing rat alveolar stem cells also includes the following steps: transferring the cell suspension to a T25 cell bottle, placing it in an incubator for culture, and replacing the culture medium every 2-3 days, characterized in that the cell incubator environment is 37°C and 5% CO2, simulating the in vivo environment of cell growth in vitro to ensure cell activity and biological characteristics.

[0014] The above-mentioned method for obtaining and culturing rat alveolar stem cells specifically comprises the following steps:

[0015] (1) Establishment of rat alveolar socket model: SD adult rats (age 6-8 weeks, male, weight about 250-300 g) were selected, and 7% chloral hydrate (0.3 mL / 100 g) + xylazine (0.03 ml / 100 g) injection was prepared and anesthetized by intraperitoneal injection. After the anesthesia took effect, the rats were fixed on the operating table, the rats' mouths were opened with a homemade mouth opener, local disinfection was performed, all the upper and lower left molars of the rats were extracted, and compression was applied to stop bleeding.

[0016] (2) Acquisition of primary DSCs: On the third day after the establishment of the rat extraction socket model, the molar extraction wound was observed to be well healed and filled with soft healing tissue with a certain shape. The tissue block was excavated, and the alveolar bone tissue that might be mixed was removed. It was placed in a culture medium containing a 2% penicillin-streptomycin-amphotericin B mixture and stored and transported at low temperature. The adherent blood was rinsed with PBS in a clean bench, and the tissue block was trimmed into a size of 1×1×1mm, transferred to a 15ml centrifuge tube, centrifuged at 800rpm for 5min, and the supernatant was discarded; 2mg / ml type I collagenase and 2mg / ml neutral proteinase II 1:1 mixture were added, and digested in a 37° constant temperature water bath for 50min, and an equal amount of complete culture medium (containing α-MEM+10% FBS+2% penicillin-streptomycin-amphotericin B) was used for neutralization, and the cells were centrifuged at 800rpm for 5min, and the supernatant was discarded; an appropriate amount of complete culture medium was added to resuspend the cells, and the cells were transferred to a T25 cell flask and cultured in a cell incubator at 37°C and 5% CO2. Replace the culture medium every 2-3 days. When the DSCs in the T25 bottle reach 70%-80% confluence, rinse twice with PBS, add 0.25% trypsin, and place in a cell incubator for digestion for 2 minutes. Observe under a microscope, when most of the cells are detached from the wall and in a suspended state, add an equal amount of complete culture medium to stop digestion, collect the cells into a 15ml centrifuge tube, centrifuge at 800rpm for 5 minutes, add complete culture medium according to the cell amount to resuspend, and place in a T25 culture bottle for continued culture.

[0017] Another object of the present invention is to provide a method for identifying the rat alveolar stem cells.

[0018] This purpose was achieved through the following measures: the third generation of rat DSCs and PDLSCs were selected, and the clone formation, proliferation ability and multidirectional differentiation ability of DSCs were compared with rat PDLSCs. First, the stem cell phenotype of PDLSCs and DSCs was identified by flow cytometry, and then the proliferation and clone formation ability of PDLSCs and DSCs were identified by CCK-8 method and crystal violet staining; finally, PDLSCs and DSCs were induced to adipogenesis and osteogenetically, and the expression of osteogenic-related genes and proteins of PDLSCs and DSCs was detected by Oil Red O staining, ALP and ARS staining, and RT-PCR and Western blot, and the multidirectional differentiation characteristics of DSCs were identified and compared.

[0019] The above-mentioned method for identifying alveolar stem cells specifically comprises the following steps:

[0020] (1) Phenotypic identification of DSCs: The third generation of rat DSCs and PDLSCs were washed twice with PBS, digested and centrifuged, and resuspended in flow cytometry washing buffer (3% FBS in PBS). The cells were counted to a cell concentration of 2×106 / mL, and dispensed into flow tubes. In a light-proof environment, antibodies were added to DSCs and PDLSCs: CD45-Pacifitc Blue, CD44-FITC, CD90-FITC, P75NTR-PE, incubated for 30 minutes, added flow washing buffer, centrifuged at 1000 rpm for 6 minutes, washed and centrifuged twice, the supernatant was discarded, PBS was added to resuspend the cells, and the cell phenotype was detected by flow cytometry.

[0021] (2) Identification of the clone-forming ability of rat DSCs and comparison with PDLSCs: Take the third-generation rat DSCs and PDLSCs, centrifuge and resuspend, count the cells, and culture them in a cell culture dish with a diameter of 10 cm at a cell density of 800 cells / dish. Change the medium every 2-3 days, during which time the cells can be seen to gradually proliferate and aggregate into clusters. After 12 days, rinse twice with PBS, add 5 ml of 4% paraformaldehyde to each culture dish, fix for 30 minutes, rinse three times with PBS, add 5 ml of 1% crystal violet to stain for 1 hour, rinse twice with pure water, observe the staining, and take macroscopic and microscopic photos. Calculate the clone-forming unit and clone-forming rate. Calculation formula: clone-forming rate = 100% × clone-forming number / cell inoculation number.

[0022] (3) Identification of the proliferation capacity of rat DSCs and comparison with PDLSCs: The third generation of rat DSCs and PDLSCs were routinely digested, the cells were counted and appropriately diluted, and 3×10 cells were plated in 7 96-well plates representing different culture days. 3 Cells were seeded at a density of 100 cells / well, and the cells were replicated in 5 wells. Each well was supplemented with 100ul complete medium, and the medium was changed every 2-3 days. After every 24 hours, the corresponding 96-well plate was taken out, 10ul CCK-8 was added under light-proof conditions, and incubated in a 37°C, 5% CO2 incubator for 2 hours, transferred to an ELISA reader, and the absorbance value at 450nm was detected. This was repeated every day to obtain a 7-day continuous cell proliferation curve of PDLSCs and DSCs.

[0023] (4) Identification of the multidirectional differentiation characteristics of rat DSCs and comparison with PDLSCs: The third generation of rat DSCs and PDLSCs were selected and cultured at 2×10 5 Cells were plated at a concentration of 100 cells / mL in a 6-well plate and cultured in a 37°C, 5% CO2 incubator. After 48 hours, when the cell fusion rate reached 80%, the original culture medium was discarded and replaced with adipogenic induction medium and osteogenic induction medium, respectively. After 7 days of osteogenic induction, ALP staining was performed; RT-PCR and Western blot were used to detect the expression of osteogenic-related genes and proteins ALP and RUNX2; after 21 days of osteogenic induction and adipogenic induction, ARS staining and Oil Red O staining were performed to identify the adipogenic and osteogenic differentiation abilities of rat alveolar stem cells.

[0024] Beneficial Effects

[0025] (1) The rat DSCs obtained by the method of the present invention have a long spindle-shaped and polygonal cell morphology, good growth activity and proliferation potential, and stable clone formation ability. The proliferation activity and clone formation ability of rat DSCs are better than those of rat PDLSCs.

[0026] (2) The rat DSCs obtained by the method of the present invention highly express the mesenchymal stem cell phenotype P75NTR, CD90, and CD44, and lowly express the MSCs negative phenotype CD45; P75NTR, CD90, CD44, and CD45 are used to identify the characteristics of mesenchymal stem cells. (3) The rat DSCs obtained by the method of the present invention can be induced to differentiate into adipocytes and osteoblasts in vitro, demonstrating their multipotency as mesenchymal stem cells. It was also verified that the osteogenic differentiation ability of rat DSCs is superior to that of rat PDLSCs. (4) The process of obtaining rat DSCs by the method of the present invention provides a better understanding of the molecular dynamic events occurring during the healing process of the alveolar socket. The early healing tissue in the alveolar socket after tooth extraction contains a large number of bioactive factors, such as CXCLI2, OPN, BMP, etc., which regulate the expression of genes and transcription factors and have an important influence on the stemness of DSCs. MSCs from the bone marrow, epithelium, adipose tissue and muscle around bone damage in the body rely on each other, which further explains the complementary effects of multiple factors in the healing process of the alveolar socket and promotes the healing of the alveolar socket, and helps promote research in the field of the healing mechanism of the alveolar socket and dental implants.

[0027] (5) The rat DSCs obtained by the present invention showed superior proliferation activity, clone formation ability and osteogenic differentiation ability to rat PDLSCs, indicating that DSCs can be used as a source of seed cells. It was also verified that the best time to obtain rat DSCs is the third day after tooth extraction, and the healing alveolar socket is an ideal tissue for extracting dental mesenchymal stem cells, which has important research value in the field of periodontal regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Normal rat molars and central incisors

[0029] Figure 2 HE staining of alveolar socket sections at 0, 1, 3, and 7 days after tooth extraction in rats: On the 3rd day after tooth extraction, a small amount of red blood cells were seen accumulating in the alveolar socket, and the alveolar socket was replaced by fibrous connective tissue.

[0030] Figure 3 P75NTR immunohistochemistry in the alveolar socket model of rats on days 0, 1, 3, and 7 after tooth extraction: On day 3 after tooth extraction, the DAB staining-positive expression areas were mainly scattered around the fibrous connective tissue in the center of the alveolar socket, and a small amount of staining was visible on the inner wall of the alveolar socket.

[0031] Figure 4 Gross photos of the rat upper left molar alveolar socket immediately after the model was established and on the 3rd day: On the 3rd day, the molar extraction wound healed well and was filled with healing tissue with a soft texture and a certain shape.

[0032] Figure 5 Primary and subcultured PDLSCs (100×)

[0033] Figure 6 DSCs primary and subculture (100×): The cells are spindle-shaped or irregular in shape with tentacles, with abundant cytoplasm and gradually proliferate into clusters.

[0034] Figure 7 Flow cytometry identification of PDLSCs results

[0035] Figure 8 The results of DSCs identification by flow cytometry showed that DSCs highly expressed MSCs-related phenotypes P75NTR, CD90, and CD44, and lowly expressed MSCs-negative phenotype CD45.

[0036] Fig. 9 Cell clone formation of PDLSCs and DSCs (40×): DSCs formed more cell clusters with larger area than PDLSCs.

[0037] Table 10 The cloning rate of DSCs and PDLSCs (%) (x±S, n=3): The cloning rate of DSCs was 29.65%±1.05%, and the cloning rate of PDLSCs was 19.21%±1.36%, and the difference was statistically significant (P<0.001).

[0038] Fig.10 Comparison of clone formation rates between DSCs and PDLSCs

[0039] Table 11 The proliferation difference between DSCs and PDLSCs detected by CCK-8 (x±S, n=3): On the fifth, sixth and seventh days, the proliferation ability of DSCs was higher than that of PDLSCs, and the difference was statistically significant (P<0.05).

[0040] Fig.11 CCK-8 assay for cell proliferation

[0041] Fig.12 Oil Red O staining (100×): Vacuole-like structures were observed in the cytoplasm of DSCs and PDLSCs, and scattered red-stained lipid droplets of varying sizes were observed.

[0042] Fig.13 ALP staining (100×): DSCs were stained more densely and darker than PDLSCs.

[0043] Fig.14 Comparison of Alizarin Red staining (100×): The number of mineralized nodules in DSCs is greater than that in PDLSCs.

[0044] Table 15 Comparison of mRNA expression levels of ALP and RUNX2 in DSCs and PDLSCs (x±S, n=3): The expression of osteogenesis-related genes ALP and RUNX2 in both DSCs and PDLSCs was upregulated, and the mRNA expression level in DSCs was higher than that in PDLSCs, and the difference was statistically significant (P<0.05).

[0045] Fig.15 RT-PCR detection of osteogenesis-related gene expression (P<0.05)

[0046] Fig.16 Western Blot detection of osteogenesis-related protein expression

[0047] Table 17 Comparison of protein expression levels of ALP and RUNX2 in two groups of cells (x±S, n=3)

[0048] Fig.17 Quantitative gray value expression: The expression levels of ALP and RUNX2 in DSCs were higher than those in PDLSCs, and the difference was statistically significant (P < 0.05)

[0049] The present invention is further described in conjunction with embodiments and drawings.

[0050] Example 1

[0051] Histological and immunohistochemical analysis of the rat alveolar socket model to verify the optimal time point for obtaining DSCs

[0052] Adult SD rats (6-8 weeks old, male, weighing about 250-300 g) were selected. 7% chloral hydrate (0.3 mL / 100 g) + xylazine (0.03 ml / 100 g) injection was prepared and anesthetized by intraperitoneal injection. After the anesthesia took effect, the rats were fixed on the operating table. A homemade mouth opener was used to open the rats' mouths. After local disinfection, all the upper and lower left molars of the rats were extracted ( Figure 1 -A) and extraction of rat central incisors ( Figure 1 -B). Apply pressure to stop bleeding.

[0053] On the 0th, 1st, 3rd and 7th days after the rat alveolar socket model was established, one rat was taken each and killed by overdose of anesthetic. The modeled left mandible was dissected out, rinsed with running water for 5 minutes, fixed with 4% paraformaldehyde tissue fixative for 1 day, and then immersed in 10% EDTA decalcification solution. The decalcification solution was changed every 3 days. After about one and a half months, the mandibular tissue specimen could be easily pierced with a needle and the decalcification was stopped.

[0054] Take anhydrous ethanol and use pure water to prepare 75%, 85% and 95% ethanol solutions. The decalcified mandibular tissue was dehydrated in a dehydrator, with 75% alcohol for 1 hour, 85% alcohol I for 50 minutes, 95% alcohol II for 50 minutes, 100% alcohol I for 50 minutes, 100% alcohol II for 50 minutes, 100% alcohol and xylene 1:1 mixture for 20 minutes, xylene I for 25 minutes, xylene II for 25 minutes, paraffin I for 1 hour, paraffin II for 2 hours, paraffin III for 3 hours. After embedding, slice the tissue.

[0055] Paraffin sections were dewaxed in xylene Ⅰ and xylene Ⅱ for 10 minutes, reacted in anhydrous ethanol Ⅰ, anhydrous ethanol Ⅱ, 95% alcohol, 85% alcohol, and 75% alcohol for 5 minutes, soaked in ultrapure water for 5 minutes, stained with hematoxylin for 5 minutes, rinsed with tap water for 3 minutes, hydrochloric acid alcohol differentiation solution for 5 seconds, rinsed with tap water for 3 minutes, 85% alcohol for 3-5 minutes, eosin staining for 3-5 minutes, rinsed with tap water for 5 seconds, dehydrated with gradient alcohol, transparentized with xylene, and sealed with neutral gum. The changes in the morphology of the alveolar socket tissue at different days after tooth extraction were observed. The results showed that the situation immediately after tooth extraction was similar to that on the first day after tooth extraction. A large number of red blood cells were seen in the alveolar socket, and a small number of osteoclasts were seen at the edge of the bone ( Figure 2 -A, B, C, D); On the third day after tooth extraction, a small amount of red blood cells were seen in the alveolar socket, and the alveolar socket was replaced by fibrous connective tissue ( Figure 2 -E,F); On the 7th day after tooth extraction, new bone cells were observed in the alveolar socket. The number of new bone cells increased but the arrangement was disordered, forming a large area of ​​bone callus. The surrounding fibrous connective tissue proliferation and a large number of osteoclasts were observed ( Figure 2 -G,H).

[0056] Paraffin sections were dewaxed in xylene I and xylene II for 10 min, reacted in anhydrous ethanol I, anhydrous ethanol II, 95% alcohol, 85% alcohol, and 75% alcohol for 5 min; rinsed with distilled water, immersed in citrate buffer (PH6.0), heated at high temperature in a microwave oven for 10 min, stopped for 8 min, heated at medium-high temperature for 10 min, and washed with PBS three times, each time for 5 min; placed in 3% hydrogen peroxide solution, reacted for 10 min, washed with PBS three times, each time for 5 min; dripped normal goat serum for 20 min, and added primary antibody; kept at 4°C overnight, washed with PBS three times the next day, each time for 5 min, and added secondary antibody; kept at 37°C for 30 min, washed with PBS three times, each time for 5 min; added DAB color developing solution, rinsed with distilled water, stained with hematoxylin for 5 min, rinsed with tap water, dehydrated with gradient alcohol, treated with xylene, sealed with neutral gum for preservation, and the colored area of ​​the target indicator was observed under a microscope. The results showed that the situation immediately after tooth extraction was similar to that on the first day after tooth extraction. P75NTR expression was observed on the inner wall of the alveolar socket and the edge of the bone marrow foramen, but no color was observed in the center of the alveolar socket ( Figure 3 -A, B, C, D). On the third day after tooth extraction, the DAB staining positive expression area was mainly scattered around the fibrous connective tissue in the center of the alveolar socket, and a small amount of staining was also seen on the inner wall of the alveolar socket ( Figure 3 -E,F); 7 days after tooth extraction, P75NTR was expressed in the fibrous connective tissue at the edge of the new trabeculae ( Figure 3 -G,H).

[0057] Example 2

[0058] Acquisition and culture of primary rat DSCs cells

[0059] All upper and lower left molars of SD adult rats were extracted and pressure was applied to stop bleeding ( Figure 4 -A). After enzymatic digestion of periodontal ligament tissue, cells crawled out after about 3 days. The cells were spindle-shaped or polygonal and gradually proliferated into clusters ( Figure 5 ).

[0060] On the third day after the rat tooth extraction socket model was established, the tooth extraction wound healed well and was filled with soft healing tissue with a certain shape ( Figure 4-B), dig out the tissue block, pay attention to remove the alveolar bone tissue that may be mixed, store it in a culture medium containing 2% penicillin-streptomycin-amphotericin B, transport it at low temperature, rinse the blood attached to ESEHT with PBS in a clean bench, trim the tissue block into 1×1×1mm size, transfer it to a 15ml centrifuge tube for centrifugation, centrifuge it at 800rpm for 5min, add a 1:1 mixture of 2mg / ml type I collagenase and 2mg / ml neutral proteinase II, place it in a 37° constant temperature water bath for digestion for 50min, and neutralize it with an equal amount of complete culture medium containing α-MEM+10% FBS+2% penicillin-streptomycin-amphotericin B. After centrifugation, add an appropriate amount of complete culture medium, resuspend the tissue block, transfer it to a T25 cell bottle, and culture it in a cell incubator at 37℃ and 5% CO2. After 3 days, cells crawl out of the adherent tissue block. The cells are spindle-shaped or irregular in shape with tentacles. The cells are plump in clathrin and gradually proliferate into clusters ( Figure 6 -A). The first medium change is 4-6 days later. Cells are observed crawling out under the microscope. The medium is changed every 2-3 days. When the cell growth fusion rate reaches about 80%, the cells are washed twice with PBS, and 0.25% trypsin is added. The cells are placed in a 37°C, 5% CO2 incubator for digestion for 2 minutes. When the cells fall off and become round under the microscope, an equal amount of complete medium can be added to stop digestion. The cells are centrifuged at 800 rpm for 5 minutes, and complete medium is added to continue culturing (6-B).

[0061] Example 3

[0062] Identification of stem cell phenotypes in rat DSCs

[0063] DSCs and PDLSCs that were routinely passaged to the third generation were washed twice with PBS, digested and centrifuged, and resuspended in flow cytometry washing buffer (PBS with 3% FBS) to a cell concentration of 2x10 6 / mL, and dispensed into pre-labeled flow tubes. In a light-proof environment, antibodies were added to DSCs: CD45-Pacifitc Blue, CD44-FITC, CD90-FITC, P75NTR-PE, incubated for 30 minutes, washed with PBS containing 3% FBS, centrifuged at 1000rpm for 6 minutes, washed twice, centrifuged at 1000rpm for 6 minutes, discarded the supernatant, resuspended with appropriate amount of PBS, and checked by flow cytometry. Flow cytometry identification showed that the third-generation DSCs and PDLSCs highly expressed the MSCs-related phenotypes P75NTR, CD90, and CD44, and low-expressed the MSCs-negative phenotype CD45 ( Figure 7 , Figure 8 ), which indicates that DSCs and PDLSCs are the sources of MSCs.

[0064] The third generation DSCs and PDLSCs were centrifuged and resuspended, and the cells were counted and placed in a 10 cm diameter cell culture dish at 800 cells / dish. The medium was changed every 2-3 days. After 12 days, PBS was rinsed twice, 5 ml of 4% paraformaldehyde was added to each culture dish, fixed for 30 minutes, rinsed 3 times with PBS, 5 ml of 1% crystal violet was added for staining for 1 hour, and washed twice with pure water. The staining was observed, and the macroscopic and microscopic images were taken. DSCs formed more cell clusters with considerable area ( Fig. 9 ), the clone formation rate of DSCs and PDLSCs was calculated, and the results showed that the clone formation rate of DSCs was higher than that of PDLSCs, and the difference was statistically significant (P<0.001). Fig.10 , Table 10)

[0065] DSCs and PDLSCs of the third generation were selected, routinely digested, counted and appropriately diluted, and plated at 3 × 10 in 7 96-well plates representing different culture days. 3 Cells were seeded at a density of 100 cells / well, and 5 replicate wells were made for each cell type. Each well was supplemented with 100ul of complete medium, and the medium was changed every 2-3 days. The corresponding 96-well plate was taken out every 24 hours, 10ul of CCK-8 was added under light-proof conditions, and incubated in a 37°C, 5% CO2 incubator for 2 hours. The plate was transferred to an ELISA reader and the absorbance at 450nm was detected. This was repeated every day to obtain a 7-day continuous cell proliferation curve of PDLSCs and DSCs ( Fig.11 ), the data analysis results showed that on the fifth, sixth and seventh days, the proliferation ability of DSCs was higher than that of PDLSCs, and the difference was statistically significant (P < 0.05) (Table 11).

[0066] The above results suggest that the rat alveolar stem cells obtained and cultured by this method have the characteristics of mesenchymal stem cell origin, good growth activity and proliferation potential, stable clone formation ability, and their proliferation activity and clone formation ability are better than periodontal ligament stem cells.

[0067] Example 4

[0068] Multidirectional differentiation potential of rat DSCs

[0069] Adipogenic induction: PDLSCs and DSCs of the third generation were selected and counted at 2×10 5The cells were plated in a 6-well plate at a density of 100 cells / well. Induction solutions A and B were prepared according to the instructions of the kit. After 48 hours, when the cell fusion rate reached 80%, the cells were rinsed with PBS twice and replaced with adipogenic induction medium. The cells were induced for 21 days, during which the A and B induction solutions were replaced alternately. After the formation of lipid droplets of appropriate size and sufficient number under the microscope, the cells were gently rinsed with PBS 2-3 times, fixed with 4% paraformaldehyde for 30 minutes, and the Oil Red O staining working solution was prepared. 2 ml of staining solution was added to each well, and the cells were stained at room temperature for 30 minutes. The residual staining solution was rinsed with PBS, and 1 ml of pure water was added to the wells. The cells were observed and photographed under a microscope. The results showed that vacuolar structures were visible in the cytoplasm of the two groups of cells, and scattered red-stained lipid droplets of varying sizes ( Fig.12 ).

[0070] Osteogenesis induction: The third generation PDLSCs and DSCs were selected, washed twice with PBS, and then diluted into PDLSCs and DSCs cell suspension after conventional digestion and centrifugation. 5 The cells were inoculated at a density of 100 cells / well in a 6-well plate. After culturing for 48 hours, the cells were observed to grow to a confluence of about 80%, and then replaced with osteogenic induction medium (α-MEM medium containing 10mmol / L β-glycerophosphate sodium, 50μg / ml vitamin C, 1×10-8mol / L dexamethasone, 1% penicillin-streptomycin-amphotericin B mixture, and 10% FBS). The medium was changed every 2-3 days, and the test was performed after 7 and 21 days of osteogenesis. On the 7th day of osteogenic induction, the old culture medium in the plate was discarded, and the cells were rinsed twice with PBS. 2ml of 4% paraformaldehyde was added to each well and fixed for 30 minutes. The staining solution was prepared according to the ratio of A solution: B solution = 2:1 in the BCIP / NBT alkaline phosphatase staining solution color development kit. The cells were stained for 30 minutes, rinsed with pure water 3 times, photographed with a stereo microscope, and then placed under an inverted microscope to observe the staining. The results showed that the DSCs group was more intensively stained and the color was darker ( Fig.13 ). On the 21st day of osteogenic induction, both cell types were in good condition under the microscope, with the appearance of scattered round high-density calcium nodules. The cells were rinsed with PBS three times, fixed with 4% paraformaldehyde for 30 minutes, and 2 ml of 0.2% Alizarin Red S staining solution was added to each well. The cells were stained at room temperature and away from light for 30 minutes, then rinsed with pure water three times. The cell staining was observed under an inverted microscope. The results showed that both groups could form mineralized nodules, and the number of nodules in the DSCs group was greater than that in the PDLSCs group ( Fig.14 ).

[0071] On the 7th day of osteogenic induction, discard the original induction solution, rinse twice with PBS, add 500ul RNA-easylsolation Reagent to the wells of the 6-well plate, and after cell lysis, collect the cell lysate and put it into a 1.5ml enzyme-free tube. Add 2 / 5 volume of DEPC water to each tube and let it stand for 5min; centrifuge at 1200rpm and 4℃ for 15min, take the upper layer of liquid containing RNA in the three layers of liquid, add an equal volume of isopropanol solution, blow and mix, let it stand for 10min; centrifuge at 11200rpm and 4℃ for 10min, and discard the supernatant. Add 500ul of 75% ethanol prepared by DEPC water and anhydrous ethanol, resuspend the precipitate, centrifuge at 9100rpm and 4℃ for 3min, discard the supernatant, let it dry at room temperature, and add appropriate amount of DEPC water to dissolve. Detect the concentration of extracted RNA. Remove the DNA that may be contained in the RNA product, prepare a mixed solution in a centrifuge tube (template RNA 1ug, 4×gDNA wiper Mix 4ul, DEPC water up to 16ul), pipette to mix, reaction conditions: 42℃, 2min. Reverse transcription cDNA synthesis: directly add 5×HiScript III qRT SuperMix 4ul to the reaction tube in step 1, reaction conditions: 37℃, 15min; 85℃, 5s.

[0072] Gene primer design and synthesis:

[0073]

[0074] RT-PCR reaction solution 20ul (Rnase Free dH2O 6ul, TB Green Premix Ex Taq III (Tli RnaseH Plus) (2×) 10ul, PCR Forward Primer 10uM 1ul, PCR Reverse Primer 10uM 1ul, cDNA 2ul). RT-PCR amplification conditions:

[0075]

[0076] After 7 days of osteogenic induction, the results showed that the expression of osteogenic-related genes ALP and RUNX2 in both groups of cells was upregulated, and the mRNA expression level in the DSCs group was higher than that in the PDLSCs group, and the difference between the two groups was statistically significant (P < 0.05) (Table 15, Fig.15 ).

[0077] Western Blot detection of osteogenic-related proteins. On the 7th day of osteogenic induction, the original culture medium was discarded, PB was washed twice, 100ul protein lysis buffer (RIPA: phosphatase inhibitor: PMSF = 100:1:1) was added to each well, and the cells were lysed at 4℃ for 30min. The cells were collected into a centrifuge tube, centrifuged at 12000rpm and 4℃ for 10min, and the supernatant containing protein was collected into a new 1.5ml EP tube. The protein concentration was determined by BCA method. The sample was added with an appropriate amount of loading buffer, denatured by boiling at 100℃ for 5min, and stored at -80℃. According to the molecular weight of the target protein, a lower gel of appropriate concentration and thickness was prepared. After mixing according to the instructions (lower gel solution: lower gel buffer = 1:1), a coagulant in the corresponding proportion was added, mixed, gel was poured, and anhydrous ethanol was added to cover the upper edge of the glass plate. After the lower separation gel solidified, the anhydrous ethanol was poured out, the filter paper was dried, and the upper gel solution was prepared. After mixing, it was poured into the glass plate and the comb teeth were inserted. The glass plate is connected to the electrophoresis tank, and the electrophoresis tank is filled with SDS-PAGE electrophoresis fluid. The comb teeth on the upper gel are pulled out, and marker 2ul is added to the holes on both sides. According to the measured protein concentration of the sample, 10ul of the sample is added to the hole, and the electrophoresis is performed at 110V for 40min. When bromophenol blue is dispersed to the bottom of the gel, the electrophoresis is terminated. Cut the PVDF membrane, soak it in methanol solution for 1min for activation, wet it layer by layer in the order of filter paper, PVDF membrane, gel and filter paper, remove the bubbles between the PVDF membrane and the gel, close the transfer clip, put it in the electrotransfer box, and transfer the membrane at 400mA for 30min. After the transfer is completed, pay attention to distinguish the front and back of the PVDF membrane, wash it with TBST 3 times, 5min each time, add 8% skim milk solution, block it at room temperature for 2h, and wash the PVDF membrane with TBST 3 times, 5-10min each time. According to the instructions, put the membrane into the prepared primary antibody dilution solution, put it in a 4℃ refrigerator, and incubate it overnight on a shaker. The next day, wash the PVDF membrane with TBST three times, 10 minutes each time. Then put the membrane into the prepared secondary antibody dilution solution and incubate it at room temperature for 2 hours. Take out the membrane and wash it with TBST three times, 10 minutes each time. Dry the membrane with filter paper, prepare ECL exposure working solution in equal proportion, add it on the membrane, make the exposure solution completely and evenly cover the membrane, let it stand for 2 minutes, develop it on the machine and collect data ( Fig.16 ). The WB quantitative gray value showed that the expression levels of ALP and RUNX2 in the DSCs group were higher than those in the PDLSCs group, and the difference between the two groups was statistically significant (P<0.05)( Fig.17 , Table 17).

[0078] In summary, DSCs extracted from the early healing tissue of the alveolar socket of SD rats have the characteristics of mesenchymal stem cells and differentiate into adipocytes and osteoblasts in a specific in vitro induction solution. It is also verified that the proliferation activity, clone formation ability and osteogenic differentiation ability of rat DSCs are superior to those of rat PDLSCs. DSCs can be used as potential seed cells for periodontal tissue regeneration and repair.

Claims

1. A method for culturing rat alveolar stem cells, characterized in that: Histological staining and immunohistochemical staining were performed on the rat alveolar socket model, and it was determined that the best time to obtain rat DSCs was the third day after the rat alveolar socket model was established.

2. The method for culturing rat alveolar stem cells according to claim 1, characterized in that: The optimal time point for obtaining rat DSCs was determined based on the expression of P75NTR in the alveolar socket after tooth extraction in rats.

3. A method for culturing rat alveolar stem cells as claimed in claims 1 and 2, wherein an early alveolar healing tissue block is obtained, centrifuged and digested, and the tissue is dispersed into a cell suspension, characterized in that: Enzymatic digestion of tissue blocks for 50 minutes successfully digested tissue fragments and digested cell aggregates to obtain a suspension.

4. A method for culturing rat alveolar stem cells as described in claim 3, further comprising centrifuging at 800 rpm for 5 minutes after enzyme digestion, discarding the supernatant, and resuspending the cells.

5. A method for culturing rat alveolar stem cells as described in claim 4, wherein the primary cells are cultured in a cell incubator at 37°C and 5% CO2, and the culture medium is replaced every 2-3 days.

6. A method for culturing rat alveolar stem cells as described in claim 1, comprising the following steps: (1) Establishment of rat alveolar socket model: SD adult rats (age 6-8 weeks, male, weight about 250-300 g) were selected, and 7% chloral hydrate (0.3 mL / 100 g) + xylazine (0.03 ml / 100 g) injection was prepared and intraperitoneally injected to anesthetize the rats. The rats were fixed on the operating table, and the rats' mouths were opened with a homemade mouth opener. Local disinfection was performed, and all the upper and lower left molars of the rats were extracted, and pressure was applied to stop bleeding. (2) Determine the optimal time point for obtaining rat alveolar stem cells: On the 0th, 1st, 3rd, and 7th days after the rat alveolar socket model was established, one rat was killed by anesthesia, and the left mandible of the modeled rat was dissected and made into paraffin sections for HE staining and immunohistochemical staining to observe the formation of alveolar socket healing tissue and the expression of P75NTR. The HE staining results showed that immediately after tooth extraction and on the first day after tooth extraction, a large number of red blood cells were accumulated in the alveolar socket, and a small number of osteoclasts were seen at the bone edge; on the third day after tooth extraction, a small number of red blood cells were accumulated in the alveolar socket, and the alveolar socket was replaced by fibrous connective tissue; on the seventh day after tooth extraction, new bone cells were formed in the alveolar socket, the number increased and the arrangement was disordered, forming a large area of ​​bone callus, and fibrous connective tissue proliferation and a large number of osteoclasts were seen around it. Immunohistochemistry results showed that P75NTR expression was observed on the inner wall of the alveolar socket and the edge of the bone marrow foramen immediately after tooth extraction and on the first day after tooth extraction, but no staining was observed in the center of the alveolar socket; on the third day after tooth extraction, the DAB staining-positive expression areas were mainly scattered around the fibrous connective tissue in the center of the alveolar socket, and a small amount of staining was also observed on the inner wall of the alveolar socket; on the seventh day after tooth extraction, P75NTR was expressed in the fibrous connective tissue at the edge of the new trabeculae. (3) Acquisition and culture of primary rat alveolar stem cells: On the third day after the establishment of the rat extraction socket model, the molar extraction wound was observed to be well healed and filled with soft healing tissue with a certain shape. The tissue block was excavated, and the alveolar bone tissue that might be mixed was removed. It was stored in a culture medium containing a 2% penicillin-streptomycin-amphotericin B mixture and transported at low temperature. Rinse the adhered blood with PBS in the clean bench, trim the tissue block into 1×1×1mm size, transfer it to a 15ml centrifuge tube, centrifuge it at 800rpm for 5min, and discard the supernatant; add 2mg / ml type I collagenase and 2mg / ml neutral proteinase II 1:1 mixture, digest it in a 37° constant temperature water bath for 50min, neutralize it with an equal amount of complete culture medium (containing α-MEM+10% FBS+2% penicillin-streptomycin-amphotericin B), centrifuge it at 800rpm for 5min, and discard the supernatant; add an appropriate amount of complete culture medium to resuspend the cells, transfer them to a T25 cell bottle, and culture them in a cell incubator at 37℃ and 5% CO2. Change the culture medium every 2-3 days. When the DSCs in the T25 bottle reach 70%-80% confluence, rinse them with PBS twice, add 0.25% trypsin, and place them in a cell incubator for digestion for 2min. Observe under a microscope. When most of the cells are detached from the cell wall and are in a suspended state, add an equal amount of complete culture medium to stop digestion. Collect the cells into a 15 ml centrifuge tube and centrifuge at 800 rpm for 5 min. Add complete culture medium according to the cell amount to resuspend and place in a T25 culture flask for continued culture.

7. A method for identifying rat alveolar stem cells: first, use CD45, CD44, CD90, and P75NTR to identify the cell phenotype of DSCs; use CCK-8 method and crystal violet staining to identify and compare the proliferation and clone formation ability of PDLSCs and DSCs; induce adipogenesis and osteogenesis of PDLSCs and DSCs, respectively, use Oil Red O staining, ALP and ARS staining, and use RT-PCR and Western blot to detect the expression of osteogenic-related genes and proteins of PDLSCs and DSCs, and identify and compare the multidirectional differentiation characteristics of DSCs.

8. A method for identifying rat alveolar stem cells as described in claim 7, comprising the following steps: (1) Phenotypic identification of rat DSCs and PDLSCs: The third generation DSCs and PDLSCs were washed twice with PBS, digested and centrifuged, and resuspended in flow cytometry washing buffer (3% FBS in PBS). The cells were counted to a cell concentration of 2×10 6 / mL, and dispensed into flow tubes. In a light-proof environment, antibodies were added to DSCs and PDLSCs: CD45-Pacifitc Blue, CD44-FITC, CD90-FITC, P75NTR-PE, incubated for 30 minutes, added flow washing buffer, centrifuged at 1000 rpm for 6 minutes, washed and centrifuged twice, the supernatant was discarded, PBS was added to resuspend the cells, and the cell phenotype was detected by flow cytometry. (2) Identification of the clone-forming ability of rat DSCs and comparison with PDLSCs: Take the third-generation DSCs and PDLSCs, centrifuge and resuspend, count the cells, and culture them in a cell culture dish with a diameter of 10 cm at a cell density of 800 cells / dish. Change the medium every 2-3 days, during which time the cells can be seen to gradually proliferate and aggregate into clusters. After 12 days, rinse with PBS twice, add 5 ml of 4% paraformaldehyde to each culture dish, fix for 30 minutes, rinse with PBS three times, add 5 ml of 1% crystal violet to stain for 1 hour, rinse with pure water twice, observe the staining, and take macroscopic and microscopic photos. Calculate the clone-forming unit and clone-forming rate. Calculation formula: clone-forming rate = 100% × clone-forming number / cell inoculation number. (3) Identification of the proliferation capacity of rat DSCs and comparison with PDLSCs: The third generation DSCs and PDLSCs were routinely digested, the cells were counted and appropriately diluted, and 3×10 cells were plated in 7 96-well plates representing different culture days. 3 Cells were seeded at a density of 100 cells / well, and the cells were replicated in 5 wells. Each well was supplemented with 100ul complete medium, and the medium was changed every 2-3 days. After every 24 hours, the corresponding 96-well plate was taken out, 10ul CCK-8 was added under light-proof conditions, and incubated in a 37°C, 5% CO2 incubator for 2 hours, transferred to an ELISA reader, and the absorbance value at 450nm was detected. This was repeated every day to obtain a 7-day continuous cell proliferation curve of PDLSCs and DSCs. (4) Identification of the multidirectional differentiation characteristics of rat DSCs and comparison with PDLSCs: DSCs and PDLSCs of the third generation were selected and cultured at 2×10 5 Cells were plated at a concentration of 100 cells / mL in a 6-well plate and cultured in a 37°C, 5% CO2 incubator. After 48 hours, when the cell fusion rate reached 80%, the original culture medium was discarded and replaced with adipogenic induction medium and osteogenic induction medium, respectively. After 7 days of osteogenic induction, ALP staining was performed; RT-PCR and Western blot were used to detect the expression of osteogenic-related genes and proteins ALP and RUNX2; after 21 days of osteogenic induction and adipogenic induction, ARS staining and Oil Red O staining were performed to identify the adipogenic and osteogenic differentiation abilities of rat alveolar stem cells.

Images